Bipolar disorder (BP) poses a significant disease burden on society, due to our lack of knowledge of its etiology and mechanism of action of drugs that are used to treat it. Genetic, clinical, and epidemiological evidence suggests the presence of a complex interplay of genetic and environmental risk factors in bipolar disorder. In particular, the HPA-axis system responsible for mounting the fight-or-flight response may constitute a significant environmental factor that plays a role in precipitating and exacerbating bipolar disorder symptoms. In order to demonstrate the role of HPA-axis genes in bipolar disorder, we propose to examine in careful detail a set of genes that are: directly involved in mediating the stress-response; implicated in neuropsychiatric disorders or behavioral response to stress; or targeted by or directly influence glucocorticoid-signaling. Our proposal draws strength from preliminary data that show alterations in DNA methylation and expression of the stress-response gene FKBP5 in postmortem brains of bipolar patients. In Aim 1, we will first obtain postmortem brain tissues from control and bipolar samples (N=25 each) that have been well defined in terms of demographic information and drug history. We will process these brain tissues by fluorescence-activated cell sorting (FACS), in order to obtain a more homogeneous neuronal fraction. We will extract genomic DNA from the neuronal fractions, and, using the Methyl-Seq platform, enrich for candidate HPA-axis genes that have been implicated in mood disorders and stress-induced behavioral deficits in animal models. Enriched DNA will then be bisulfite-converted and subjected to next-generation sequencing. DNA methylation data for the candidate HPA-axis genes will be analyzed to identify differentially methylated regions (DMRs) between BP and control tissues. Further, DMRs will be independently verified using pyrosequencing, considered the gold standard for DNA methylation analysis. In Aim 2, we will extract messenger RNA from unsorted tissues to assess expression levels of the candidate HPA-axis genes, using multiple Taqman probes to target isoform-specific exon-intron junctions. We will also perform Western blotting on proteins extracted from the brain tissues to determine the concordance between gene expression and protein levels in bipolar vs. control samples. Results from Aims 1 and 2 will be further analyzed to identify potential DNA methylation-independent mechanisms, alternative splicing, and post-translational modification events specific to bipolar disorder. Taken together, the proposed experiments seek to demonstrate the role that HPA-axis genes may play in bipolar disorder pathology and to provide additional avenues for future investigation into differential regulation of HPA-axis substrates in bipolar disorder.